Opioid receptors, at both mRNA and protein levels, are expressed in a temporally and spatially specific manner, suggesting a specific control for their gene expression. It was first hypothesized that the control of the cell (tissue) type- and developmental stage-specificity of the mu opioid receptor (MOR) and the opioid receptor (DOR) is mediated by specific cis-acting DNA sequences of the gene regulatory regions, which interact with various specific regulatory protein (trans-acting) factors. Our previous proposal attempted to examine this hypothesis by first identifying (dissecting) the basic DNA elements responsible for their promoter activities and potential trans-acting factors interacting with these DNA elements. With these basic regulatory elements dissected and identified, it is further hypothesized that manifestation of these regulatory sequences and protein factors may even utilizes a higher order control which involves changes in chromatin structure of the gene pr moter of MOR and DOR. There are three specific aims in this renewal proposal: first, to continue our ungoing studies focused on detailed identification of more components (both cis- and trans-acting factors) for the MOR and the DOR gene regulation, and second to take one step further for the understanding of how these regulatory components interact with each other or with chromatin modification and nucleosome organization of MOR and DOR gene regulatory region, in order to address the physiological relevance of the regulatory components identified from Aim 1. The third aim will extend these in vitro studies into animals by using transgenic mouse models. Therefore, studies proposed in this renewal grant will utilize a combination of tissue culture systems where MOR and DOR genes are known to be active, such as NS2OY (for DOR) and P19 (for MOR) cells, and transgenic mouse systems where whole animal opioid pharmacology can be examined. It is our ultimate goal to understand the underlying mechanisms for controlling MOR and DOR gene expression, and how these regulatory pathways may interact with exogenous or environmental factors, such as hormones and drugs, to achieve a homeostatic control for MOR and DOR expression in the context of animal physiology.